Blood clotting involves a complicated cascade of events to signal the platelets and fibrin to form the clot. Thrombosis is the formation of blood clot (thrombus) inside a blood vessel, obstructing the flow of blood. Embolism occurs when a blood clot, migrates from one part of the body usually through the circulatory system and causes a blockage or occlusion in another part of the body. The occlusion can also occur at the site of thrombus formation without any migration. A thromboembolic disorder refers both to thrombosis and its principal complication which is embolization (migration of thrombus and occlusion).
At the cellular level, thrombus formation is characterized by rapid conformational changes leading to activation of blood platelets and of various plasma proproteins. In response to a range of triggering stimuli and cascading events, zymogenic prothrombin is catalyzed to thrombin. In turn, thrombin acts upon the soluble structure protein fibrinogen, cleaving the N-terminal A and B polypeptides from the alpha and beta chains to form fibrin monomer. Cleavage results in redistribution of charge density and exposure of two polymerization sites, enabling growth of the monomer into an insoluble, three dimensional polymeric network. Concurrently, thrombin acts to induce significant physiological change to a “resting” or inactive blood platelet by changing its shape. This is associated with thromboxane A2 synthesis and release of ADP from intraplatelet storage granules which enhances platelet aggregation. Such activated platelets play a dual role in hemostasis:                a) They are more adhesive and capable of binding fibrinogen and von Willebrand factor. Activated platelets adhere to subendothelial von Willebrand factor via the GPIb receptor and co-aggregate with fibrinogen and von Willebrand factor via the GPIIb-IIIa.        b) Activated platelets act as a catalytic surface for thrombin generation from its plasma pro-enzymes. This results in the formation of insoluble fibrin intermeshed within and around the platelet thrombus. This three dimensional platelet plug under pathophysiological conditions can serve to compromise circulatory system patency leading to stroke, tissue infarction, and necrosis.        
Thrombus formation can be pathogenic and is a causative factor in ischemic heart disease (myocardial infarction, unstable angina), ischemic stroke, deep vein thrombosis, pulmonary embolism, and related conditions. Vascular disease can result in hypercoagulable states, resulting in thrombus formation. Consequences of ischemic stroke include loss of function of the affected region and death.
Appearance of atherosclerotic plaques within the coronary arteries is the precursor to ischemic heart disease (IHD). Causative factors for ischemic stroke include cardiogenic emboli, atherosclerotic emboli, and penetrating artery disease. Migration of the embolus through the aorta into the carotids can result in closure of a cerebral vessel. Pulmonary embolism results from the migration of the embolus from a formation site within the deep veins of the extremities into the pulmonary vasculature. In the event of an acute blockage, consequences include rapid death by heart failure. Formation of thrombi within the deep veins of the lower extremities is characterized as deep vein thrombosis. Causative factors include blood stasis. Certain surgical procedures also correlate strongly with postoperative venous clot formation.
Stroke, a thromboembolic disorder, is the second largest cause of death in the world. Platelets play a major role in this process as first demonstrated by the beneficial effect of aspirin on stroke development or reoccurrence. There are 2 major forms of occlusive stroke formation: (a) Release of ruptured plaque and fibrin thrombi from a stenosed carotid; (b) atrial fibrillation leading to cardiac thrombus embolization. Both mechanisms are associated with distal vessel post ischemic occlusion (reperfusion injury) secondary to deposition of platelets and fibrin on anoxia-induced activated endothelial cells (Zhang et al., “Dynamic Platelet Accumulation at the Site of the Occluded Middle Cerebral Artery and in Downstream Microvessels is Associated with Loss of Microvascular Integrity After Embolic Middle Cerebral Artery Occlusion,” Brain Res 912:181-94 (2001); Choudhri et al., “Reduced Microvascular Thrombosis and Improved Outcome in Acute Murine Stroke by Inhibiting GPIIb/IIIa Receptor-mediated Platelet Aggregation,” J Clin Invest 102:1301-10 (1998); del Zoppo, “The Role of Platelets in Ischemic Stroke,” Neurology 51:S9-14 (1998)). During high shear forces, platelets adhere weakly to endothelium via conformationally-induced Von Willebrand Factor (VWF) binding to the platelet GPIb-V-IX receptor. This is followed by inside-out intracellular platelet signaling which activates the conformation of the major platelet Integrin receptor, αIIbβ3 (GPIIb-GPIIIa). This forms a more permanent, stable platelet aggregate (via fibrinogen bridging of GPIIb-IIIa receptor) on adjacent platelets. These activated platelets generate thrombin on their surface which leads to deposition of fibrin fibers within and outside of the thrombus.
Ischemic post-infusion platelet aggregation has been described to occur 1 hr after initial occlusion to as long as 23 hrs after reperfusion, both indirectly by 111In-labelled platelet deposition (Choudhri et al., “Reduced Microvascular Thrombosis and Improved Outcome in Acute Murine Stroke by Inhibiting GPIIb/IIIa Receptor-mediated Platelet Aggregation,” J Clin Invest 102:1301-10 (1998)) as well as scanning electron microscopy of cerebral vessels distal to the initial lesion (Zhang et al., “Dynamic Platelet Accumulation at the Site of the Occluded Middle Cerebral Artery and in Downstream Microvessels is Associated with Loss of Microvascular Integrity After Embolic Middle Cerebral Artery Occlusion,” Brain Res 912:181-94 (2001)).
Therapeutic lysis of pathogenic thrombi (thrombolysis) is achieved by administering thrombolytic agents. Benefits of thrombolytic therapy include rapid lysis of the thromboembolic disorder and restoration of normal circulatory function. Currently, there are mainly three thrombolytic strategies used clinically. First, inhibition of thrombin by either the indirect thrombin inhibitor heparin or direct thrombin inhibitors, such as hirudin (Meyer et al., “Local Delivery of r-Hirudin by a Double-balloon Perfusion Catheter Prevents Mural Thrombosis and Minimizes Platelet Deposition after Angioplasty,” Circulation 90:2474-2480 (1994); Topol et al., “Recombinant Hirudin for Unstable Angina Pectoris: A Multicenter, Randomized Angiographic Trial,” Circulation 89:1557-1566 (1994); Cannon et al., “Hirudin: Initial Results in Acute Myocardial Infarction, Unstable Angina and Angioplasty,” J Am Coll Cardiol 25:30 S-37S (1995)). Second, administration of Integrin αIIbβ3 (platelet glycoprotein GPIIb/IIIa) antagonists such as abciximab, tirofiban, and eptifibatide that affect the attachment of platelet to fibrinogen by mimicking RGD binding site (Tam et al., “Abciximab (ReoPro chimeric 7E3 Fab) Demonstrates Equivalent Affinity and Functional Blockade of Glycoprotein IIb/IIIa and Alpha(V) Beta3 Integrins,” Circulation 98:1085-1091 (1998); Lefkovits, “Platelet Glycoprotein IIb/IIIa Receptor Antagonists in Coronary Artery Disease,” Eur. Heart. J. 17:9-18 (1996); Scarborough et al., “Platelet Glycoprotein IIb/IIIa Antagonists: What are the Relevant Issues Concerning their Pharmacology and Clinical Use?”Circulation 100:437-444 (1999)). Third, using blood clot-dissolving agents such as TPA, streptokinase, and urokinase to make thrombolytics effective in dissolving fibrin around thrombi (Topol et al., “A Multi-center, Randomized, Placebo-controlled Trial of a New Form of Intravenous Recombinant Tissue-type Plasminogen Activator (Activase) in Acute Myocardial Infarction,” J Am Coll Cardiol 9:1205-1213 (1987); Marler et al., “Stroke: tPA and the Clinic,” Science 301:1677 (2003); Collen, “Towards Improved Thrombolytic Therapy,” Lancet 342: 34-36 (1993)). Although the above treatments have been partially successfully, some of the treated patients suffer from bleeding complications, such as hemorrhagic stroke (Sakharov et al., “Superficial Accumulation of Plasminogen During Plasma Clot Lysis,” Circulation 92: 1883-1890 (1995)). Complications include internal and external bleeding due to lysis of physiologic clots, and stroke resulting in cerebral hemorrhage.
For example, TPA is associated with secondary toxicity, such as hypofibrinogenemia and bleeding. Also, successful application of thrombolytics in ischemic stroke has not been realized. Current treatment of occlusive stroke with TPA, an agent which must be given within 3 hrs of occlusion (Su et al., “Activation of PDGF-CC by Tissue Plasminogen Activator Impairs Blood-brain Barrier Integrity During Ischemic Stroke,” Nat Med 14:731-737 (2008); Choi et al., “Endovascular Recanalization Therapy in Acute Ischemic Stroke,” Stroke 37:419-24 (2006)), is operationally feasible in ˜10% of stroke patients. A later infusion runs the risk of cerebral hemorrhage.
Animal stroke experiments with anti-platelet GPIIb-IIIa agents have successfully diminished brain infarct formation as well as permanent neurological damage (Choudhri et al., “Reduced Microvascular Thrombosis and Improved Outcome in Acute Murine Stroke by Inhibiting GPIIb/IIIa Receptor-mediated Platelet Aggregation,” J Clin Invest 102:1301-10 (1998); Abumiya et al., “Integrin α(IIb)β(3) Inhibitor Preserves Microvascular Patency in Experimental Acute Focal Cerebral Ischemia,” Stroke 31:1402-09 (2000)). However, this has been associated with increased cerebral hemorrhage and death, since antibodies against GPIIb-IIIa inhibit platelet function and induce thrombocytopenia. The development of a different approach to inhibit arterial bleeding and associated thromboembolic disorders by lysis of platelet thrombus with specific anti-GPIIIa(49-66) antibodies would be of significant clinical value. A recent clinical study on the role of Abciximab (anti-GPIIB-IIIa) in stroke was discontinued because of its high rate of hemorrhage as well as ineffectiveness (Adams et al., “Emergency Administration of Abciximab for Treatment of Patients with Acute Ischemic Stroke: Results of an International Phase III Trial: Abciximab in Emergency Treatment of Stroke Trial (AbESTT-II),” Stroke 39:87-99 (2008)).
The present invention is directed to overcoming these and other deficiencies in the art.